Early lung cancer detection by DNA methylation phenotyping of sputum-derived cells

ABSTRACT

In certain embodiments, this application discloses methods for detecting lung cancer. The method includes characterization of cells extracted from human sputum, which is a valuable tissue surrogate and source of upper respiratory cells that become cancerous early in the process of lung cancer development. The method includes the staining of extracted cells with fluorescent reporters that produce a specific pattern in the nuclei of labeled cells, which can be made visible by light microscopy. The pattern is relevant to a type of epigenetic coding of DNA known as DNA methylation, which changes in specific cells of the lung during cancer development, in comparison to normal respiratory cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/507,668, filed Feb. 28, 2017, which is a National Phase ofInternational Application No. PCT/US2015/047567, filed Aug. 28, 2015,which designated the U.S. and that International Application waspublished under PCT Article 21(2) in English, which claims priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/043,346,filed Aug. 28, 2014, each of which is hereby incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to the diagnosis, prognosis, andtreatment of cancer, and especially lung cancer.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art, or relevant to thepresently claimed invention.

Traditional methods of screening for lung cancer includemediastinoscopy, and radiographic methods, such as computed tomography(CT) and positron emission tomography (PET). Unfortunately, thesemethods are expensive and/or require exposing patients to potentiallyharmful ionizing radiation. In addition, scans are not reliable fordetecting early-stage lung cancer that may be too small to detect byradiographic methods, but nonetheless pose significant danger to apatient. This is especially relevant, because early-stage lung cancerdetection is associated with a much more favourable prognosis thanlate-stage detection.

There is clearly a need in the art for a safe, relatively inexpensive,and sensitive method for detecting lung-cancer, especially at an earlystage.

SUMMARY OF THE INVENTION

In various embodiments, the invention teaches a method for determiningif a cell is cancerous or precancerous, including: determining a global5-methylcytosine (5mC) content and/or spatial nuclear co-distribution of5mC and global DNA (gDNA) in a nucleus of the cell; and determining thatthe cell is cancerous or precancerous if the global 5mC content and/orspatial nuclear co-distribution of 5mC and gDNA in the nucleus of thecell is significantly different from a non-cancerous or non-precancerousreference cell and/or a non-cancerous or non-precancerous reference cellpopulation, or determining that the cell is not cancerous or notprecancerous if the global nuclear 5mC content and/or spatial nuclearco-distribution of 5mC and gDNA are not significantly different fromthose of a non-cancerous or non-precancerous reference cell and/or anon-cancerous or non-precancerous reference cell population. In someembodiments, the cell is determined to be cancerous or precancerous ifthe global 5mC content is significantly lower than the non-cancerous ornon-precancerous reference cell and/or non-cancerous or non-precancerousreference cell population. In certain embodiments, the cell is obtainedfrom a biological sample. In some embodiments, the biological sampleincludes sputum. In certain embodiments, the sputum includes respiratorycells. In certain embodiments, the cancerous cell or precancerous cellis of lung cancer origin. In some embodiments, the biological sample isobtained from a subject who has a history of smoking cigarettes. In someembodiments, the biological sample is obtained from a subject who doesnot have a history of smoking cigarettes. In some embodiments, thebiological sample is obtained from a subject who has lung cancer and hasnot been treated for lung cancer. In certain embodiments, the biologicalsample is obtained from a subject who has received a lung cancertreatment selected from the group consisting of: radiation therapy,chemotherapy, surgery, and combinations thereof. In some embodiments,global 5mC and gDNA contents are determined with a microscope after thecell has been subjected to (a) immunofluorescence staining with anantibody specific for 5mC, and (b) counterstaining with4′,6-diamidino-2-phenylindole (DAPI). In certain embodiments, spatialnuclear co-distribution of 5mC and gDNA is determined with a microscopeafter the cell has been subjected to (a) immunofluorescence stainingwith an antibody specific for 5mC, and (b) counterstaining with4′,6-diamidino-2-phenylindole (DAPI). In some embodiments, the sputumsample was obtained from a subject by a method that includesadministering hypertonic saline into the subject's respiratory tract;and collecting a quantity of sputum that is expelled from the subject asthe result of inhaling said hypertonic saline. In some embodiments, thehypertonic saline is administered via a nebulizer. In certainembodiments the hypertonic saline is 3-5% NaCl. In certain embodiments,the microscope is a confocal scanning microscope with a resolution equalto or less than 500 nanometers.

In various embodiments, the invention teaches a method that includesobtaining a biological sample from a subject, wherein the biologicalsample includes a cell; determining a global 5-methylcytosine (5mC)content and/or spatial nuclear codistribution of 5mC and global DNA(gDNA) in a nucleus of the cell; determining that the cell is cancerousor precancerous if the global 5mC content and/or spatial nuclearcodistribution of 5mC and gDNA in the nucleus of the cell issignificantly different from a non-cancerous or non-precancerousreference cell and/or non-cancerous or non-precancerous cell population;and determining that the subject has a high risk of developingclinically verifiable cancer, if it is determined that the cell iscancerous or precancerous. In some embodiments, the method also includestreating the subject for cancer, if it is determined that the subjecthas a high risk for developing clinically verifiable cancer, or if it isdetermined that the subject has developed clinically verifiable cancer.In some embodiments, the biological sample includes sputum. In someembodiments, the sputum includes respiratory cells. In some embodiments,the cancerous cell or precancerous cell is of lung cancer origin. Incertain embodiments, the subject has a history of smoking cigarettes. Insome embodiments, the subject does not have a history of smokingcigarettes.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in the referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 demonstrates, in accordance with an embodiment of the invention,the workflow of 3D quantitative DNA Methylation Imaging (3D-qDMI)includes three steps: (1) cytological specimen preparation/staining, (2)3D-imaging of specimens, and (3) computational image/data analysis forspecimen characterization.

FIG. 2 demonstrates, in accordance with an embodiment of the invention,workflow of 3-D image analysis (example shown with DU145 human prostatecancer cells). Confocal 2D image stacks from the two channels of5-methylcytosine (5mC) and 4′,6-diamidino-2-phenylindole (DAPI) areloaded. DAPI represents global nuclear DNA (gDNA). Extracted nuclear5mC/DAPI patterns are displayed as 2D density scatter plots ofvoxel-intensities of the two channels. Example patterns are shown fortwo selected nuclei. The 5mC/DAPI codistribution pattern of the entirepopulation is created through superposition of patterns from allindividual nuclei that could be distinct in appearance/statisticsrepresenting highly differential codistribution of nuclear 5mC and DAPIsignals. Units indicated on the axes of the scatter plots are ArbitraryIntensity Units.

FIG. 3 demonstrates, in accordance with an embodiment of the invention,diagnostic output of 3D-qDMI (Left): characterization of the sputum cellpopulation based on 5mC (green) and gDNA/DAPI (blue) texture features(DNA methylation phenotypes) in the fluorescence image. Cells can becategorized into different similarity degrees by “soft-qualifiers” thatspan increasing value ranges associated with color codes (Right). The3D-qDMI software uses this coding to convert the original fluorescenceimage (across all confocal image layers) into a color map and acorresponding tabular display for better visualization andinterpretation of the resulting data. The data leads to identificationand enumeration of the different types of cells for determining theheterogeneity of DNA methylation phenotypes in cell populations.

FIG. 4 demonstrates, in accordance with an embodiment of the invention,normal parenchyma and the humoral region of a fluorescently labeledsection from a newly diagnosed, surgically resected lung cancer. Cellnuclei (blue) in normal lobules (A) and magnified boxed subarea (B) showhigher degree of DNA methylation (5mC, green) compared with severelyhypomethylated nuclei in ductal regions of the tumor (C) and magnifiedboxed subarea (D) on the same section; cytokeratin 8 (red) was used as amarker to delineate the epithelial compartments.

FIGS. 5A & 5B demonstrate, in accordance with an embodiment of theinvention, global DNA methylation phenotyping of cells and tissues with3D-qDMI. The method was able to successfully distinguish between thedifferent cell types based on differential 5mC/DAPI distributionpatterns (scatter plots): calculated and displayed as individual heatmap scatter plots (DAPI=x-axis, 5mC=y-axis) for the entire cellpopulation as the reference plot, as well as for each nucleus as shownfor the selected nuclei N1 and N2 for each cell category. Non-small celllung cancer (NSCLC) cell lines A549 and H157 display a reduction inglobal 5mC compared to immortalized epithelial respiratory cells(BEAS-2B). H157 cells, which are reported to have more metastaticpotential than A549 cells, are even more hypomethylated (flatter curve).The same comparative relation can be found in surgically removed tissuefrom a lung cancer patient and adjacent normal lung tissue and also frommatching sputum samples of the lung cancer patient versus the healthyperson (with no cancer): The cytometric 5mC/DAPI signatures found inhealthy sputum cells are very similar to the patterns seen in cells inthe phenotypically normal area (see FIG. 3 for a real image) and BEAS-2Bcells. In contrast, severe hypomethylation can be observed in a smallnumber of sputum cells (N2-type)—in the background of a larger number ofcells with normal 5mC-phenotype (N1-type)—of the cancer patient thatmatches signatures of cells from the tumoral region and the moreaggressive H157 cell line (higher metastatic potential). In other words,the rare sputum cells with aberrant 5mC-phenotype have a strongresemblance with well-characterized aggressive cancer cells (in tumorsand tumor-derived cell lines). The regression line (yellow-dashed) andthe upper and lower signal borderlines ML1 and ML2 are characteristicand determine the four angles α, β, γ, and δ for each prototypic celltype. The resulting factor F=[(α/γ)×(β/δ)] is specific to each celltype. All cell populations show high homogeneity: i.e. high degree of5mC-phenotype similarity between cells, as judged by the respectivecategory-maps, and the similarity between the scatter plots ofindividual nuclei (N1 and N2) compared to the plot of the respectiveentire population. Units indicated on the axes of the scatter plots inFIGS. 5A and 5B are Arbitrary Intensity Units.

FIG. 6 demonstrates, in accordance with an embodiment of the invention,a bright field microscopic image of relatively flat human epithelialcells, derived from induced sputum. (A) Cells were isolated frommucus-liquid fraction of sputum and captured on a glass slide usingculturing techniques. A few milliliters of sputum can contain hundredsto thousands of cells. (B) Magnification of an area reveals the relativesubstructure of layered cells that are mononuclear.

FIG. 7 demonstrates, in accordance with an embodiment of the invention,confocal images of fluorescently labeled sputum-derived human cells. Thecytoplasm is delineated by the epithelial-cell marker cytokeratin 19(CK19, in red), cell nuclei are delineated by DAPI (in blue), and globalnuclear DNA methylation is visualized by an antibody specific to 5mC (ingreen). The sputum of healthy individuals (control) contains anoverwhelming majority of highly methylated cells (type 1, left column)and sporadically a few CK19-positive hypomethylated cells (type 2, leftcolumn). It is assumed that the hypomethylation is facultative to theearly stage after cell division. In contrast, the sputum of a lungcancer patient additionally contains a significant number of round cellswith almost no cytoplasm (type 2, right column). These cells areCD34/CD45-negative, indicating that they are not of hematopoietic and/orleukocytic nature. The respective nuclear 5mC/DAPI codistributionpatterns, presented as scatter plots, show that normally methylated(type 1) cells in both sputum-donor groups display a steep regressionline (∂>45°), whereas hypomethylated cells (type 2) produce a muchflatter regression line (∂<<45°). Moreover, a typical signature of thelung cancer-specific rounded cells is the much less dispersed and narrowco-distribution of 5mC and DAPI. Units indicated on the axes of thescatter plots are Arbitrary Intensity Units.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Allen et al., Remington: The Science and Practice of Pharmacy22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al.,Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology 3rd ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006);Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7^(th) ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton,Dictionary of DNA and Genome Technology 3^(rd) ed., Wiley-Blackwell(Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A LaboratoryManual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N.Y. 2012), provide one skilled in the art with a general guide to manyof the terms used in the present application. For references on how toprepare antibodies, see Greenfield, Antibodies A Laboratory Manual2^(nd) ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013);Köhler and Milstein, Derivation of specific antibody-producing tissueculture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July,6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No.5,585,089 (1996 December); and Riechmann et al., Reshaping humanantibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, certain terms are defined below.

“Conditions” and “disease conditions,” as used herein, may include butare in no way limited to those conditions that are associated withcancer or pre-cancer, including, but in no way limited to lung cancer,cancer of the head or neck, cancer of the upper aerodigestive tract,cervical cancer, ovarian cancer, urethral cancer, bladder cancer, andcolorectal cancer.

“Mammal,” as used herein, refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domesticated mammals, such asdogs and cats; laboratory animals including rodents such as mice, ratsand guinea pigs, and the like. The term does not denote a particular ageor sex. Thus, adult and newborn subjects, whether male or female, areintended to be included within the scope of this term. While cancer orprecancer can be detected in humans according to the inventive methodsdescribed herein, detecting cancer in any mammal according to theinventive methods is within the scope of the invention.

The terms “global 5mC” and “5mC content” are used hereininterchangeably, and in each case can be defined as the total amount of5-methylcytosine molecules present in a cell nucleus.

The term “global DNA (gDNA)” as used herein means the total amount ofDNA present in a cell nucleus.

The term “clinically verifiable cancer” as used herein means cancer thatis verifiable by traditional means of cancer detection, including butnot limited to minimally-invasive mediastinoscopy, noninvasiveradiographic methods, such as computed tomography (CT), positronemission tomography (PET), magnetic resonance imaging (MM), and thelike.

By way of additional background, it is becoming more and more evidentthat epigenetic mechanisms such as DNA methylation have a stronginfluence in the development of multi-cellular systems, in their healthymaintenance and in their structural and functional decline during agingand at an accelerated rate by diseases such as cancer, alongside withand even without the coexistence of genetic mutations. Therefore,methods for measuring DNA methylation are vital in understanding thesemechanisms in efforts for combating cancers and securing healthy aging.There is no doubt that imaging, alongside with molecular techniques, isplaying an indispensable role in the differential quantification of DNAmethylation in cells and tissues.

Measuring changes in DNA methylation is valuable, since it correlateswith early events in carcinogenesis and tumor progression, and can serveas a signature in early diagnostics and therapeutic monitoring. In thissense, the inventors' approach, as described in certain embodimentsherein, to apply quantitative DNA methylation imaging for earlydetection of lung cancer revives the idea of in situ measuringepigenetic features such as DNA methylation in exfoliated respiratorycells for their characterization, for a cell-by-cell based pathologicaldiagnosis.

DNA methylation imaging, which was introduced for tissuecharacterization towards the end of the 1990s did not gain muchpopularity in comparison to contemporaneously developed molecularmethods, including PCR-based, array-based, sequencing, high-pressureliquid chromatography (HPLC), and mass spectrometry, for two reasons:(i) it was applied in combination with radio-labeled or enzymaticreporters for detection, which either lack sensitivity, multiplexingcapability or affect repeatability/consistency of the assay, and (ii)did not provide enough significance in differential results due to lowimage resolution. Enormous improvements in high-resolution imaging andcomputational capacity within recent years have been supportive to thedevelopment of more sophisticated tools in cell-based assays that can beapplied to biomedical research and clinical diagnosis. This was also apre-requisite for the development of 3D-qDMI to revisit the concept ofnondestructive imaging of large-scale changes on the higher-orderchromatin structure by epigenetic reporters such global DNA methylation.

In short, the 3D-qDMI approach described herein is especiallyadvantageous because it allows for (1) high-resolution imaging of5-methylcytosine (5mC) and global DNA (gDNA), and (2) digital extractionof three 5mC-relevant features as diagnostic signatures for early lungcancer detection: (i) the 5mC load (content), (ii) the spatial nuclearcodistribution of 5mC and gDNA, and (iii) measurement of cell-populationheterogeneity based on the first two 5mC features, in order tocharacterize respiratory epithelial cells in sputum samples (FIG. 2 ).

Compared to current molecular approaches and a few previouslow-resolution imaging-based attempts that either average 5mCmeasurements across a large population of cells or only measure mean 5mCintensity values in cell nuclei, 3D-qDMI leverages the extraction ofdifferential 5mC-relevant information by considering secondary effectsof DNA methylation imbalances that occur throughout cellulartransformation, especially hypomethylation of gDNA. In particular thelatter mechanism elicits reorganization of the genome within cellnuclei, affecting nuclear architecture. This phenomenon is welldescribed in basic cell biological research, but has not yet beenexploited well in cancer pathology. The image analysis applied in someembodiments of the inventive method covers this gap and displays therelevant changes as intensity distribution of the two types of signalsthat reflect said phenomena: (a) 5mC-signals created throughimmunofluorescence targeting and (b) gDNA represented by DAPI-signalsthat are generated by subsequent counter-staining of the same cells, asDAPI intercalates into AT-rich DNA the main component of highlyrepetitive and compact heterochromatic sequences. Overall, the methodresults in images that represent maps of sputum cells with a spectrum ofdifferential DNA methylation phenotypes (5mC/DAPI texture features) thatcorrelate with cell morphology (epithelial and mesenchymal cellphenotypes) and growth behavior (high-proliferative cancer cells,moderately growing normal cells, and growth-arrested senescent cells).

Although lung cancer cells are one type of cancer cells that could bedetected according to the methods described herein, analysis of 5mCcontent and/or 5mC and gDNA spatial nuclear co-distribution could beused to detect any cancer cell.

With the foregoing additional background in mind, certain specificnon-limiting embodiments are described below.

In various embodiments, the invention teaches a method for determiningif a cell is cancerous or precancerous. In some embodiments, the methodcomprises, consists of, or consists essentially of determining the5-methylcytosine (5mC) content and/or spatial nuclear co-distribution of5mC and global DNA (gDNA) in a nucleus of the cell; and determining thatthe cell is cancerous or precancerous if the 5mC content and/or spatialnuclear co-distribution of 5mC and gDNA in the nucleus of the cell issignificantly different from a non-cancerous or non-precancerousreference cell and/or a non-cancerous or non-precancerous reference cellpopulation, or determining that the cell is not cancerous or notprecancerous if the 5mC content and/or spatial nuclear co-distributionof 5mC and gDNA are not significantly different from those of anon-cancerous or non-precancerous reference cell and/or a non-cancerousor non-precancerous reference cell population. In this context, asignificant difference is defined as equal to or higher than 25% in 5mCcontent, and/or equal to or higher than 20 degrees in the angle of theregression line (also called trendline) herein referred to as or 6 or 0,whereby cancerous or pre-cancerous cells exhibit less 5mC content and/ora smaller regression-line angle of the 5mC/DAPI colocalization scatterplot, compared to a reference non-cancerous or non-precancerous cell ornon-cancerous or non-precancerous population of cells: when theDAPI-values define the x-axis and the 5mC values define the y-axis. Insome embodiments, 25-99%, or 30-80%, or 40-60% difference in 5mC contentis significant. In some embodiments, 20-90 degrees, or 30-80 degrees, or40-70 degrees, or 50-60 degrees in the angle of the regression line issignificant. In certain embodiments, the cell is determined to becancerous or precancerous if the 5mC content is significantly lower thanthe non-cancerous or non-precancerous reference cell and/ornon-cancerous or non-precancerous reference cell population. In certainembodiments, the cell is obtained from a biological sample. In someembodiments, the biological sample includes sputum. In certainembodiments, the sputum includes respiratory cells. In some embodiments,the origin of the cancerous cell or precancerous cell is of the upperaerodigestive tract, which includes a cell associated with anyanatomical structure or set of structures in the path from the lungs tothe lips or nares of the nose. This may include, but is in no waylimited to cells of the lungs, trachea, esophagus, mouth, nose, andsinuses. In some embodiments, the cancerous cell or precancerous cell isof lung cancer origin. In some embodiments, the cancerous cell orprecancerous cell is of a lung tumor origin. In some embodiments thecancerous cell or precancerous cell is of an esophageal origin. Incertain embodiments, the biological sample is obtained from a subjectwho has a history of smoking cigarettes. In some embodiments, thebiological sample is obtained from a subject who does not have a historyof smoking cigarettes. In various embodiments, the biological sample isobtained from a subject who has received a lung cancer treatment thatmay include, but is in no way limited to radiation therapy,chemotherapy, surgery, and combinations thereof. In some embodiments,the biological sample is obtained from a subject who has not receivedlung cancer treatment. In certain embodiments, the global 5mC and gDNAcontents of individual cell nuclei, as well as the spatial nuclearco-distribution of 5mC and gDNA are determined with a microscope afterthe cell has been subjected to (a) immunofluorescence staining with anantibody specific for 5mC, and (b) counterstaining with4′,6-diamidino-2-phenylindole (DAPI).

Any commercially available monoclonal antibody specific for 5mC could beutilized in conjunction with the inventive methods described herein. Forexample, the 5mC antibody could be obtained from vendors such as AvivaSystems Biology, Corp. (San Diego, Calif.), GeneTex, Inc. (Irvine,Calif.), Active Motif, Inc. (Carslbad, Calif.), and Diagenode, Inc.(Denville, N.J.) to name a few. In some embodiments, the 5mC antibody isthe antibody described in Reynaud C, Bruno C, Boullanger P, Grange J,Barbesti S, Niveleau A. Monitoring of urinary excretion of modifiednucleosides in cancer patients using a set of six monoclonal antibodies.Cancer Lett 1992 Mar. 31; 63(1):81, which is hereby incorporated hereinby reference in its entirety as though fully set forth.

In certain embodiments the phenotypes of individual sputum-derived cellsis determined with a microscope after the cells have been subjected toimmunofluorescence staining with antibodies against cell-type specificmarkers. These include but are not restricted to antibodies specific forcytokeratins and cell surface molecules.

In some embodiments, the sputum sample described above was obtained froma subject by a method including administering hypertonic saline into thesubject's respiratory tract; and collecting a quantity of sputum that isexpelled from the subject as the result of inhaling said hypertonicsaline. In certain embodiments, the hypertonic saline is administeredvia an ultrasonic nebulizer or a non-ultrasonic nebulizer. In someembodiments, the hypertonic saline is 3-5% NaCl.

In embodiments of the invention in which visualization and/orquantification of 5mC content, gDNA, and/or spatial nuclearco-distribution of 5mC and gDNA is required, these features may bevisualized and/or quantified through the use of optical imaging systemssuch as widefield epifluorescence microscopes and scanners, confocalmicroscopes and scanners, multi-photon microscopes and scanners, andsuper-resolution microscopes (nanoscopes) and scanners, as well ascombinatorial modalities thereof. In some embodiments, a microscope isused for this visualization and/or quantification. In certainembodiments, the microscope is a confocal scanning microscope. In someembodiments, the confocal scanning microscope has a lateral resolution(in x- and y-axes) in the range of 100-200 nm and a vertical resolution(in z-axis) of approximately 500 nm

In various embodiments, the invention teaches a method that comprises,consists of, or consists essentially of obtaining a biological samplefrom a subject, wherein the biological sample includes a cell;determining a 5-methylcytosine (5mC) content and/or spatial nuclearco-distribution of 5mC and global DNA (gDNA) in a nucleus of the cell;determining that the cell is cancerous or precancerous if the 5mCcontent and/or spatial nuclear co-distribution of 5mC and gDNA in thenucleus of the cell is significantly different from a non-cancerous ornon-precancerous reference cell and/or non-cancerous or non-precancerouscell population; and treating the subject for cancer according to anymethod described herein if it is determined that the cell is cancerousor precancerous. In some embodiments, if a subject is determined to havea cancerous or precancerous condition, then the subject is monitored fordisease progression, rather than implementing treatment. In someembodiments, the cancerous cell or precancerous cell originates in theaerodigestive tract, as described herein. In certain embodiments, thebiological sample includes sputum. In some embodiments, the sputumincludes respiratory cells. In certain embodiments, the cancerous cellor precancerous cell is of lung cancer origin. In some embodiments, thesubject has been previously treated for cancer, including any cancertype described herein. In some embodiments, the subject has not beenpreviously treated for cancer, including any cancer type describedherein. In some embodiments, the subject has a history of smokingcigarettes. In certain embodiments, the subject does not have a historyof smoking cigarettes.

In various embodiments, the invention teaches a method for determiningthe presence or absence of a cancerous cell or a precancerous cell in abiological sample that includes a plurality of cells. In someembodiments, the method includes: utilizing high-resolution imaging todetermine 5-methylcytosine (5mC) load/content and/or spatial nuclearco-distribution of 5mC and global DNA (gDNA) for each of a plurality ofcells in the biological sample; and optionally determining cellpopulation heterogeneity for the plurality of cells based on said MeCload and spatial nuclear co-distribution of 5mC and gDNA. In certainembodiments, it is determined that a cancerous cell or precancerous cellis present in the biological sample if 5mC load and/or spatial nuclearco-distribution of 5mC and gDNA in any cell in the biological sample issignificantly different from a non-cancerous or non-precancerousreference cell population and/or any cell in the biological sample issignificantly different with respect to 5mC load and/or spatial nuclearco-distribution of 5mC and gDNA compared to the global pattern of theentire population of cells visualized in the biological sample. In someembodiments, it is determined that a cancerous cell or precancerous cellis not present in the biological sample if the 5mC load and/or spatialnuclear co-distribution of 5mC and gDNA are not significantly differentfrom those of a non-cancerous or non-precancerous reference cellpopulation and/or no cell in the biological sample is significantlydifferent with respect to 5mC load and/or spatial nuclearco-distribution of 5mC and gDNA compared to the global pattern of theentire cell population in the biological sample. In some embodiments,the biological sample includes sputum. In certain embodiments, thesputum includes respiratory cells. In some embodiments, the cancerouscell or precancerous cell detected/determined is associated with lungcancer. In certain embodiments, the cancerous cell or precancerous celldetected/determined is associated with non-small cell lung cancer(NSCLC). In certain embodiments, the method can be used to diagnose asubject with lung cancer, including NSCLC, at any stage, on the basis ofthe presence of a cancerous cell in the biological sample. In certainembodiments, the sputum is obtained from a subject who has a history ofsmoking cigarettes. In some embodiments, the sputum is obtained from asubject who has received any lung cancer treatment including but in noway limited to radiation therapy, chemotherapy, surgery, andcombinations thereof. In some embodiments, the sputum is obtained from asubject who has not received one or more of the above-described cancertreatments. In some embodiments, the sputum is obtained from a subjectwho has not received treatment for cancer. In some embodiments, thesputum is obtained from an individual who has never been diagnosed withcancer. In certain embodiments, 5mC patterns are visualized afterimmunofluorescence staining with an antibody specific for 5mC. In someembodiments, gDNA is visualized after counterstaining with4′,6-diamidino-2-phenylindole (DAPI).

In certain embodiments, the invention teaches quantifying the number ofcells in the sample that have been identified as cancerous orprecancerous by implementing the foregoing testing methods, andcomparing that number of cancerous or precancerous cells to a referencenumber of cancerous or precancerous cells in individuals who havecancer, or pre-cancer, and/or comparing the tested sample with areference number of cancerous or precancerous cells in individuals whodo not have cancer, or pre-cancer.

In certain embodiments, the inventive methods described herein includeobtaining the sputum sample from the subject. In some embodiments, thesputum sample is obtained by administering hypertonic saline into thesubject's respiratory tract; and collecting a quantity of sputum that isexpelled from the subject as the result of inhaling said hypertonicsaline. In certain embodiments, the hypertonic saline is administeredvia an ultrasonic nebulizer. In some embodiments, the hypertonic salineis about 3 to 5% NaCl. In some embodiments, the ultrasonic nebulizer hasan output of about 1 to 2 mL/minute. In some embodiments the salinesolution is inhaled for a period of about 5 to 20 minutes. In somealternative embodiments, the sputum sample is obtained by using ahandheld nebulizer to dispense hypertonic saline into the subject'srespiratory tract. In some embodiments, the hypertonic saline is withina range of NaCl described above. In some embodiments, the hypertonicsaline is dispensed for a period of time within a range described above.

While the administration of hypertonic saline is one method of inducingsputum, one would readily appreciate that alternative methods ofinducing sputum could also be used to obtain a sample that could be usedwith the inventive methods. Merely by way of non-limiting examples,bronchoscopy and bronchoalveolar lavage could also be used.

In various embodiments, the invention teaches a method for treating asubject who has been diagnosed with cancer or a precancerous conditionaccording to one or more of the methods described herein. In someembodiments, the method comprises, consists of, or consists essentiallyof administering chemotherapy and/or radiation therapy and/or performingsurgery to resect all or a portion of a tumor on the subject, whereinthe subject was diagnosed with cancer or a precancerous condition viaany method described herein. In some embodiments, the subject has beendiagnosed with lung cancer.

Although the foregoing methods are aimed at detecting lung cancer andpre-cancerous lesions in a subject, as indicated above it would also bepossible to utilize the same basic principles of the inventive methodsdescribed herein to analyze samples and detect cancer or pre-cancerouslesions of different origins. Merely by way of non-limiting examples,saliva and/or mucous secretions could be assayed to determine thepresence or absence of head and/or neck cancer; colon and/or rectalsecretions could be assayed to determine the presence or absence ofcolon and/or rectal cancer; cervical secretions could be assayed todetermine the presence or absence of cervical cancer; vaginal and/orcervical secretions could be assayed to determine the presence orabsence of ovarian cancer, and fluids from the urethra could be assayedto determine the presence or absence of urethral, bladder or kidneycancer.

Further, although tests involving 5mC are the primary focus of theexamples described herein, one of skill in the art would readilyappreciate that other cytosine variations, such as 3-methylcytosine,5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine couldalso be used as bases for distinguishing between cancerous (orprecancerous) and noncancerous cells, by applying essentially the samedetection and analysis methods described herein. Therefore, evaluationof any cytosine methylation, by using the methods described herein, isintended to be within the scope of the present application. Moreover,although tests described in the specific examples set forth hereininvolve DAPI as the primary dye for delineating gDNA as well as thenuclear volume, one of skill in the art would also appreciate that otherdyes, which bind double-stranded DNA in a nonsequence-specific mannerand can be used for gDNA quantification. These may include but are notlimited to propidium iodide, the Hoechst dyes (including Hoechst 33258and Hoechst 33342), ethidium bromide, SYBR Green, SYBR Gold, Pico Green,the SYTOX dyes (including SYTOX Green, SYTOX Blue, and SYTOX Orange),the SYTO dyes, the YOYO and TOTO families of dyes (including YOYO, TOTO,JOJO, BOBO, POPO, YO-PRO, and PO-PRO), as well as actinomycin D and7-aminoactinomycin D (7-AAD), which could also be used for the samepurposes.

Given the significant difference in global nuclear 5mC load anddistribution between pre-cancerous or cancerous cells and their normalcounterparts, these differences can be visualized and measured usinglight microscopy in a rapid, parallel manner at single-cell resolutionfor the characterization of thousands of cells within biologicalsamples. In some embodiments, the global nuclear content and relativedistribution of 5mC versus global gDNA (delineated by DAPI) insputum-derived cells and cell populations are analyzed. These nuclearentities are not static and reorganize during cellular transformation ofnormal healthy cells into precancerous and cancerous cells. In thiscontext, a powerful aspect of scatter plots is their ability to depictmixture models of simple relationships between variables. Theserelationships can reflect cellular patterns as specific signatures, inwhich the variables can be nuclear structures as shown in the case ofnuclear 5mC patterns versus DAPI-stained gDNA (Tajbakhsh J, Wawrowsky KA, Gertych A, Bar-Nur O, Vishnevsky E, Lindsley E H, Farkas D L).Characterization of tumor cells and stem cells by differential nuclearmethyl-ation imaging. In: Farkas D L, Nicolau D V, Leif R C, editors.Proceedings Vol. 6859 Imaging, Manipulation, and Analysis ofBiomolecules, Cells, and Tissues VI 2008. p 68590F). We have shown thatsuch reorganizations can be dynamically monitored by scatter plottingthe signal distributions of global 5mC and gDNA, with their differentialdistribution becoming visible as changes in the plotted patterns. Inother words, the 2D scatter plots represent signal frequencyco-distributions of the targeted two nuclear entities, and theco-distribution plots can be considered as cell-specific signatures (SeeTajbakhsh J, Wawrowsky K A, Gertych A, Bar-Nur O, Vishnevsky E, LindsleyE H, Farkas D L). Characterization of tumor cells and stem cells bydifferential nuclear methyl-ation imaging. In: Farkas D L, Nicolau D V,Leif R C, editors. Proceedings Vol. 6859 Imaging, Manipulation, andAnalysis of Biomolecules, Cells, and Tissues VI 2008. p 68590F); GertychA, et al. Automated quantification of DNA demethylation effects in cellsvia 3D mapping of nuclear signatures and population homogeneityassessment. Cytometry A 2009; 75:569-83; Gertych A, et al. Measuringtopology of low-intensity DNA methylation sites for high-throughputassessment of epigenetic drug-induced effects in cancer cells. Exp CellRes 2010; 316(19):3150-60; Oh J H, et al. Nuclear DNA methylation andchromatin condensation phenotypes are distinct between normallyproliferating/aging, rapidly growing/immortal, and senescent cells.Oncotarget 2013; 4:474-93. In some embodiments, these 5mC/gDNAcodistribution signatures together with the content of global 5mC andgDNA are the three parameters/biomarkers that are considered in thecharacterization of sputum-derived individual cells and cell populationsfor the identification of pre-cancerous and cancerous cells.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

EXAMPLES Example 1 Additional Background

Lung Cancer and Current State of Diagnosis

In the year 2010, there were over 200,000 new cases of lung cancerreported in the United States, accounting for 15% of all new cancercases. The estimated number of lung cancer deaths in the same period wasroughly 160,000, representing ˜28% of all cancer-related deaths.Unfortunately, due to limited treatment options, lung cancer is the mostcommon cause of cancer related mortality. If this disease is diagnosedin an early stage, a complete surgical resection of the tumor provides afavorable chance for cure. Therefore, early detection of this diseasehas been the focus of many attempts in the past few decades. Severaltrials utilizing radiographical techniques including chest X-ray, chestcomputed tomography (CT), and positron emission tomography (PET) scanshave shown mixed results with unclear clinical benefits and harbor veryhigh cost. As indicated above, radiographical methods for earlydetection of lung cancer, including chest CT scans, involve a high doseof radiation, which by itself imposes a higher risk of developingsecondary malignancies if used frequently. As a result, frequent use ofchest CT scans for screening lung cancer is probably not safe oreconomical.

Importantly, previous trials have utilized sputum cytology for earlydetection of lung cancer, but they mainly depended on evaluatingmorphological changes of exfoliated epithelial respiratory cells, andeach of them failed to detect lung cancer cases to the point that couldshow a meaningful clinical advantage.

On the other hand, assessment of methylation status of certain genes inthe sputum samples of high-risk patients has been successful in earlydetection of lung cancer lesions. Unfortunately, lung cancer is aheterogeneous group of diseases and no uniform abnormality is identifiedin all cases. Equally important, the analysis of cell samples thataverage gene methylation status across a large number of cells maydisguise the important subtle information that is specific to a smallersubgroup of cancerous cells in sputum and therefore bias the analysisresults. Therefore, relying on the abnormalities of a subset of genesacross all sputum cells to detect lung tumors probably may only cover asubgroup of cases. After considering the shortcomings of previouslyavailable diagnostic methods, the inventors sought to analyze the globalDNA methylation status of exfoliated respiratory cells in the sputum ina cell-by-cell fashion as a tool for early detection of lung cancer.

DNA Methylation in Cancer Diagnosis

The perfect epigenetic equilibrium of normal cells is substantiallyaltered when cells become transformed. The resulting epigeneticalterations at the DNA level fall into two categories: (i) gene-specifichypermethylation of CpGs in gene promoters in gene-rich genomic regionstermed CpG-islands, and (ii) genome-wide hypomethylation, a largepercentage of which occurs in repetitive DNA elements. Aberrantmethylation patterns are associated with several cancer types.Genome-wide hypomethylation parallels closely to the degree ofmalignancy and is a ubiquitous finding. The analysis of DNAhypomethylation has largely remained unexploited. Cancer cell lines,widely used as research models, exhibit a large variation in genome-widedemethylation, which reflects tissue-specificity and unlikely resultsfrom stochastic processes. A malignant cell can contain 20-60% lessgenomic methylcytosine than its normal counterpart. The loss of methylgroups is achieved mainly by hypomethylation of repetitive DNAsequences, which account for more than 90% of the human genome,including transposable elements (˜48% of genome) such as short and longinterspersed nuclear elements (SINES and LINES, respectively), largelyacquired as retroviruses throughout evolution. Global methylation isalso clinically relevant, as demonstrated by associations betweenclinical outcome and global methylation levels in a number of cancertypes. Global hypomethylation seems to be related to cancer progression,since loss of global methylation tends to become more pronounced asprecancerous lesions progress. To date, differential DNA methylationanalysis has been quantitatively assessed mostly by molecular approachesincluding electrophoretic, chromatographic, PCR-based, array-based, andsequencing technologies. Despite tremendous improvement in specificity,sensitivity, and the inherent single-base resolution of these methods,they remain technically challenging in the high-throughput analysis ofsingle cells. These include the limitation of PCR-based approaches inmultiplexing, and the challenging sensitivity and cost issues ofwhole-genome sequencing, especially for the interrogation of repetitiveelements. Alternatively, and considering the prevalence and load of DNAmethylation imbalances, especially hypomethylation of repetitiveelements, imaging-based assessment of global nuclear 5mC patternsprovides a powerful tool to simultaneously analyze and characterize alarge number of cells, as the underlying molecular processes involvelarge-scale chromatin reorganization visible by light microscopy.

Significance of Quantitative DNA Methylation Imaging

As demonstrated herein, a method of quantitative DNA methylation imaging(3D-qDMI) has been developed and applied to lung cancer. Thisnondestructive method entails the parallel quantitative measurement of5-methylcytosine load and spatial nuclear distribution, in order tocharacterize cells and tissues (See Tajbakhsh J, et al. Characterizationof tumor cells and stem cells by differential nuclear methylationimaging. In: Farkas D L, Nicolau D V, Leif R C, eds. Imaging,Manipulation, and Analysis of Biomolecules, Cells, and Tissues. SanJose, Calif.: Proceedings of the SPIE 2008; 6856:6859F1-10; Gertych A,et al. Automated quantification of DNA demethylation effects in cellsvia 3D mapping of nuclear signatures and population homogeneityassessment. Cytometry A 2009; 75:569-83; Gertych A, et al. Measuringtopology of low-intensity DNA methylation sites for high-throughputassessment of epigenetic drug-induced effects in cancer cells. Exp CellRes 2010; 316:3150-60; Gertych A, et al. Homogeneity assessment of cellpopulations for high-content screening platforms. In: InformationTechnology in Biomedicine. Vol. 2. Advances in intelligent and softcomputing, Vol. 69. Ewa Pietka and Jacek Kawa, Editors, Springer Verlag,Heidelberg, Germany; Tajbakhsh, J. et al. (2012). 3-D Quantitative DNAMethylation Imaging for Chromatin Texture Analysis inPharmacoepigenomics and Toxicoepigenomics. In Epigenomics: FromChromatin Biology to Therapeutics. K. Appasani, editor. CambridgeUniversity Press, Cambridge, United Kingdom; each of which isincorporated herein by reference in its entirety as though fully setforth). The workflow of an embodiment of 3D-qDMI is illustrated in FIG.1 .

Given the large dynamic range in 5mC load and distribution, 3D-qDMIallows for the rapid, parallel, morphometric, single-cell resolutioncharacterization of thousands of cells within heterogeneous sputumsamples. The following highlights some of the advantages of 3D-qDMIapplicable to using non-invasive surrogates such as sputum samples inlung cancer diagnostics and clinical decision-making: (i) 3D-qDMI doesnot require cellular enrichment through error-prone separation methods;(ii) the method does not require time-consuming DNA extraction and DNAamplification, (iii) 3D-qDMI provides cell-by-cell analysis; (iv) themethod enables the heterogeneity assessment of cell populations,including frequency of different cell types in regards to DNAmethylation features; (v) irrelevant cells can be identified andexcluded from analysis, which would prevent data skewing through sampleimpurity by infiltrating hematopoietic cells; (vi) the cost-efficientcytometric approach can be automated and is amenable to scale, thereforecan be easily developed and implemented in clinical settings. Thecytometric approach can be applied to simultaneous multi-colorhigh-content imaging. Hence, cells of interest and/or infiltratinghematopoietic cells can be additionally labeled for cell-specificmarkers. Subsequently, irrelevant cells can be identified in the outputdata and eliminated before data analysis. Furthermore, the method iscompatible with using microscopic slides and Society for BiomolecularSciences (SBS)-format microplates as a support for deposition ofsputum-derived cells. Therefore, the method has the potential forimplementation in the high-throughput clinical and diagnosticenvironment, that is routinely applying said formats for cancerdiagnostics. In detail, the three steps of sample preparation, staining,and scanning can be automated with existing commercially availablehigh-throughput instruments. Image and data analysis are computerizedprocesses that are naturally performed in an automated fashion, and onlylimited by computational capacity.

Analysis of Samples

The 3D-qDMI software implemented in certain embodiments described hereinwas designed to perform sophisticated 3-D image analysis of individualcells (as opposed to the collective analysis and result-output) withinan image frame, thus allowing flexible elimination and combination ofcells for variable statistics. In some embodiments, the outcome of the5mC/DAPI colocalization pattern can be represented as a scatter plot(See FIG. 2 ). However, one of skill in the art would readily appreciatethat there are many other ways to represent this type of data.

As demonstrated in FIG. 2 , 5mC features such as 5mC/DAPI colocalizationpatterns can vary between cells within a population. Therefore, cellpopulation heterogeneity assessment is a valuable feature in determiningthe composition of the cells, i.e. the degree of phenotypic variationimportant for the identification of a low number of cells that may showaberrant MeC phenotypes similar to aggressive cancer cells. Homogeneitycan be assessed by the comparison of structural similarity within anentire cell population by expressing a relationship between anindividual nuclear 5mC/DAPI pattern and the global pattern of the entirecell population, representing the sum of all individual nuclear patterns(reference pattern).

Sputum Studies The inventors explored 3D nuclear 5mC patterns in humanupper respiratory cells derived from the sputum of a healthy individual(non-smoker with no history of cancer), as well as sputum cells from alung cancer patient (smoker) and matching tissue specimen, as well asthree human cell lines. Cell lines included the immortalized normalhuman epithelial cell line (BEAS-2B), and the NSCLC lines A549 (alveolarbasal epithelial cells) and H157 (highly invasive lung carcinoma cells).FIG. 4 shows normal parenchyma and the tumoral region of a fluorescentlylabeled section from a newly diagnosed, surgically resected lung cancer.The inventors observed common global DNA methylation patterns amongsthealthy cells that significantly differ from the 5mC/DAPI patterns ofcancerous cells and abnormal sputum cells from the cancer patient (FIG.5 ) that were significantly globally hypomethylated. All populations ofthe three different cell lines and sputum-derived cells, as well as thenormal tissue, showed a high degree of homogeneity (visualized throughKL category maps) in terms of their 5mC/DAPI codistributions, displayedas scatter plots on the cell population level and for individualrepresentative nuclei.

The inventors have introduced a measureable descriptor of each cell type(FIG. 5 ): the regression line of the plot and the upper and lowersignal borderlines ML1 and ML2 are characteristic and determine the fourangles α, β, γ, and δ for each prototypic cell type. The resultingdifferential factor F=[(α/γ)×(β/δ)] is specific to each cell type: 0.54(BEAS-2B), 0.42 (A549), 0.12 (H157), 0.78 (typical normal tissue cells),0.45 (cells of normal sputum), 0.44 (majority of N1-type cells in sputumof cancer patient), 0.01 (N2-type cell in sputum of cancer patient), and0.05 (typical cancer tissue cells). This measure underlines thedifferentiating power of global methylation patterns for detection ofnormal and malignant cells. Especially the resemblance between (N2-type)cell signatures in cancer-patient sputum and typical tumor tissue cellscan play a central role in detecting abnormal cells in sputum samples,early in the process of tumorigenesis. The observations demonstrated inFIGS. 4 and 5 that 3D nuclear DNA methylation patterns serve as a novelbiomarker for the non-invasive detection of malignant cells of therespiratory tract. In some aspects, the inventive method utilizes3D-qDMI to determine differential global DNA methylation patterns ofexfoliated respiratory cells in a sputum sample of individuals withhigher risk for developing lung cancer. Specifically, each sputum cellpopulation can be characterized by the statistics of determinedF-factors that provide an estimation of the cell composition, whichcould facilitate the detection of malignant cells.

Example 2 Methods

Preparation of Cell Specimens

Sputum induction can be performed through inhalation of hypertonicsaline (3 to 5% NaCl). Utilizing a nebulizer, aerosols can be generated,with an output at 1.5 mL/min. The subjects inhale saline solutionaerosols for a period of up to 20 min. Subjects are encouraged toexpectorate sputum after mouth rinsing with tap water every 5 minutes.Exfoliated upper respiratory cells are isolated and fixed onslides/coverslips or in microplate wells. Samples that were collected ina plastic container are kept at 4° C. until processing. Samples arediluted with phosphate-buffered saline (PBS) solution, containing 0.1%dithiothreitol (DTT) commonly known as 10% sputolysin solution, and areincubated for 20 minutes before centrifugation at 300-1500×g for 5-10minutes at room temperature in order to separate cellular and fluid(mucus) phases. This process is repeated until the cell suspensionappears to be homogeneous and clear. Then, the cell pellet isresuspended in PBS, and the cells are filtered through a 40-100 μm nylonmesh (cell strainer) to remove residual mucus and debris. Subsequently,cells are centrifuged at 300-1500×g for 5-10 minutes. The cell pellet(containing all harvested cells) is resuspended in 1-2 microliters ofepithelial-cell medium, transferred onto a microscopic glass coverslip,and cultured for 16-48 hours at 37° C. and 5% CO₂ for the cells toattach to the coverslip. In some embodiments, cell counts are performedon samples centrifuged (cytospin) and the cell sample is spread on amicroscope slide/coverslip or in a microplate well. Subsequently, cellsare fixed in 4% paraformaldehyde for 15-45 minutes and are kept in PBSat 4° C. Then, characterization of fixed cells is accomplished by 3Dquantitative DNA Methylation Imaging (3D-qDMI), as described herein.

As an alternative to the airway sputum processing method describedabove, airway sputum may be processed by any method known in the art.Merely by way of example, airway sputum processing may be performedaccording to any method described or referenced in Hamid et al. EurRespir J 2002; 20 Suppl. 37, 19s-23s.

Biochemistry

Sample analysis is accomplished through the combination ofimmunofluorescence staining for visualization of overlay methylcytosinepatterns with a specific mouse monoclonal antibody (clone 33D3) against5-methylcytosine in cell nuclei, and counterstaining with4′,6-diamidino-2-phenylindole (DAPI) for delineation of global nuclearDNA. While there are numerous publicly available protocols for stainingfor visualization of 5-methylcytosine and gDNA, in some embodiments,protocols of the following references are used: Tajbakhsh J, et al.Characterization of tumor cells and stem cells by differential nuclearmethylation imaging. In: Farkas D L, Nicolau D V, Leif R C, eds.Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues.San Jose, Calif.: Proceedings of the SPIE 2008; 6856:6859F1-10; 33.Gertych A, et al. Automated quantification of DNA demethylation effectsin cells via 3D mapping of nuclear signatures and population homogeneityassessment. Cytometry A 2009; 75:569-83; Gertych A, et al. Measuringtopology of low-intensity DNA methylation sites for high-throughputassessment of epigenetic drug-induced effects in cancer cells. Exp CellRes 2010; 316:3150-60; Gertych A, et al. Homogeneity assessment of cellpopulations for high-content screening platforms. In: InformationTechnology in Biomedicine. Vol. 2. Advances in intelligent and softcomputing, Vol. 69, 2010; Gertych A, et al. 3-D DNA methylationphenotypes correlate with cytotoxicity levels in prostate and livercancer cell models. BMC Pharmacol Toxicol. 2013 Feb. 11; 14:1; TajbakhshJ, et al. Early In Vitro Differentiation of Mouse Definitive Endoderm isNot Correlated with Progressive Maturation of Nuclear DNA MethylationPatterns. PLoS ONE 2011; 6(7):e21861; Tajbakhsh J. Covisualization ofmethylcytosine, global DNA, and protein biomarkers for In Situ 3D DNAmethylation phenotyping of stem cells. Methods Mol Biol. 2013;1052:77-88; Oh J H, et al. Nuclear DNA methylation and chromatincondensation phenotypes are distinct between normallyproliferating/aging, rapidly growing/immortal, and senescent cells.Oncotarget 2013; 4:474-93; Tajbakhsh J, et al. Dynamic heterogeneity ofDNA methylation and hydroxymethylation in embryonic stem cellpopulations captured by single-cell 3D high-content analysis. Exp CellRes. 2015; 332:190-201, each of which is incorporated herein byreference in its entirety as though fully set forth). The 5mC antibodyused for staining can be as described in(wwwdotncbi.nlm.nih.gov/pubmed/?term=Reynaud%20C%5BAuthor%5D&cauthor=true&cauthor_uid=1739950)Boullanger P, Grange J, Barbesti S, Niveleau A. Monitoring of urinaryexcretion of modified nucleosides in cancer patients using a set of sixmonoclonal antibodies. Cancer Lett 1992 Mar. 31; 63(1):81. Thespecificity of the anti-5mC antibody can be confirmed using a DNAmicroarray including cytosine variants and standard control experimentsin combination with immunocytochemistry. For exclusion of hematopoieticcells and especially white blood cells (leukocytes) in downstreamanalyses, specimens can be co-immunophenotyped with anti-CD34 andanti-CD45 antibodies. In some embodiments, the inventors also useantibodies against cytokeratins such as but not limited to CK8, CK18,and CK19 to co-label for epithelial cell markers. However, malignantrespiratory cells are dispersed among a large number of normalepithelial cells (on a slide). Therefore epithelial markers may not behelpful in the distinction of normal from abnormal cells.

Immunofluorescence (IF)

The following non-limiting protocol is for convenient processing ofsputum-derived cells that are captured onto 18 mm round glass coverslips (No. 1) and processed in 12-well microplates. Reagent volumes needto be adjusted for other cell supports and reaction chambers.

Day 1

(a) Fixation of Tissue Sections

-   -   1) Sputum-derived cells are fixed in 4% Paraformaldehyde        (PFA)/PBS for 30-45 minutes at room temperature, then washed 3        times with PBS for 3-5 minutes at room temperature. Cells not        immediately processed further shall be kept in 0.002% NaN₃/PBS        at 2-8° C.        (b) Pre-IF Processing of the Cells

2) Wash cells for 5 minutes in PBS (2 ml).

3) Permeabilize cells with 0.5% Saponin/0.5% Triton X-100/PBS (5 ml) for20 min at room temperature, and wash 3 times with PBS (2 ml) for 3-5minutes at room temperature.

4) Treat cells with 100 μg/ml RNase A/PBS (0.2 ml) for 30 minutes at 37°C., and wash 3 times 3-5 minutes with PBS (2 ml) at room temperature.

5) Block tissue with 3% bovine serum albumin (BSA)/PBS (1 ml) for 30minutes at 37° C. (prior to applying the primary antibody).

(c) First Immunofluorescence

6) Incubate tissue with primary antibody or cocktail of compatibleantibodies for cell phenotyping (as example rabbit anti-CK19 polyclonalantibody, Abcam Cat. #ab15463, at 1:1000 dilution; sheep anti-CD34polyclonal antibody, R&D Systems Cat. #AF7227, at the concentration of 1μg/ml; and chicken anti-CD45, GeneTex Cat. #GTX82139 at 1:500 dilution)in 3% BSA/PBS (0.7 ml) overnight at 2-8° C.

Day 2

7) Wash cells 4 times for 5 minutes with 01% BSA/0.1% Tween20/PBS (2 ml)at room temperature.

8) Incubate tissue with secondary antibody (for example donkey anti-goatIgG (H+L)-Alexa 568, Invitrogen, A11057) at the concentration of 5 μg/mleach in 3% BSA/PBS (0.7 ml) for 1 hour at 37° C.

9) Wash tissue 4 times with 0.1% BSA/0.1% Tween20/PBS (2 ml) for 3-5minutes at room temperature, and once with 0.1% BSA/PBS (2 ml).

10) Fix tissue in 4% PFA/PBS (1 ml) for 15 min at room temperature. Washcells 3 times for 3-5 minutes with PBS.

11) Depurinate cells with 2N HCl (1 ml) for exactly 40 min at roomtemperature, and wash 3 times with PBS (2 ml) for 3-5 minutes at roomtemperature.

12) Block cells with 3% BSA/PBS (1 ml) for 30 minutes at 37 C (prior toapplying the primary antibody).

(d) Second Immunofluorescence

13) Incubate cells with primary antibody (for example mouse anti-MeC,clone 33D3 mAb, Aviva Systems Biology, Cat. #AMM99021) at theconcentration of 1-2 μg/ml in 3% BSA/PBS (0.7 ml) overnight at 2-8° C.

Day 3

14) Wash cells 4 times for 5 minutes with 01% BSA/0.1% Tween20/PBS (2ml) at room temperature, and once with 0.1% BSA/PBS (2 ml).

15) Incubate cells with secondary antibody (for example donkey antimouse Alexa488 IgG (H+L), Invitrogen A21202; and chicken anti-rabbit IgG(H+L)-Alexa 647, Invitrogen A21443) both at the concentration of 5 μg/mlin 3% BSA/PBS (0.2 ml) for 2 hours at 37° C.

16) Wash tissue 4 times with 0.1% BSA/0.1% Tween 20/PBS (5 ml) for 5minutes and once with 0.1% BSA/PBS for 5 minutes, at room temperature.

17) Incubate tissue in 5 ml of DAPI/PB S solution (warm to roomtemperature) for 20 min at room temperature, then rinse for ˜30 sec inPBS to rinse non-specific DAPI staining.

18) Take coverslip out of the microplate and let dry completely at roomtemperature or in 37° C. oven (10-30 min), in the dark.

19) Transfer 7-10 μl of mounting solution (for example Prolong-Gold,Invitrogen) onto a clean and dry glass slide section and put coverslip(with cells facing the glass slide) onto the mounting droplet (strictlyavoid air bubbles).

Confirmatory Molecular Method

A confirmatory molecular method can be performed on extracted DNA fromisolated cells (from selected sputum samples) in parallel to verify theimage-cytometrical (3D-qDMI) 5mC feature results: (i) 5mC load and (ii)hypomethylation of repetitive DNA element classes(Alu/LINE-1/Satα/Sat2)—the major causative of global DNA hypomethylationboth can be assessed by Repeat-Sequence MethyLight (See Weisenberger D Jet al. Analysis of repetitive element DNA methylation by MethyLight.Nucleic Acids Res. 2005 Dec. 2; 33(21):6823-36, which is incorporatedherein by reference in its entirety as though fully set forth). Thismethod has proven to be an accurate surrogate of high-performance liquidchromatography (HPLC) or high-performance capillary electrophoresis(HPCE) in the measurement of 5mC load, which have been conventionallyused for global 5mC content measurements. 5mC content measurementscomparatively performed by Repeat-Seq MethyLight and 3D-qDMI has yieldedvery high correlations (0.86-0.96), (See Gertych A, et al. 3-D DNAmethylation phenotypes correlate with cytotoxicity levels in prostateand liver cancer cell models. BMC Pharmacol Toxicol. 2013 Feb. 11; 14:11which is incorporated herein by reference in its entirety as thoughfully set forth).

Image Acquisition

Image acquisition can be performed by utilizing high-resolution confocalscanning microscopy. In some non-limiting embodiments, Leica'scommercial TCS SP5 X Supercontinuum microscope (Leica Microsystems) isutilized. The system provides full freedom and flexibility in excitationand emission, within the continuous range of 470 to 670 nm—in 1 nmincrements. The microscope can be coupled with a 405 nm diode laser linefor excitation of DAPI fluorescence. Serial optical sections can becollected at increments of 200-300 nm with a Plan-Apo 63X 1.4 oilimmersion lens and pinhole size 1.0 airy unit. To avoid bleed-through,the imaging of each channel can be acquired sequentially. By way ofnon-limiting example, the typical image size can be ranging from1024×1024 to 2048×2048 with a respective voxel size of around 116 nm×116nm×230.5 nm (x, y, and z axes), and resolution of 8-16 bits per pixel inall channels. The output file format can be a series of TIFF images thatcan be utilized for 3D-image analysis.

3D Image Analysis

3D image analysis can be performed by the application of a dedicatedalgorithm developed for pattern recognition and multi-parametrichigh-content analysis, as described in Gertych A, et al. Automatedquantification of DNA demethylation effects in cells via 3D mapping ofnuclear signatures and population homogeneity assessment. Cytometry A2009; 75:569-83; Gertych A, et al. Measuring topology of low-intensityDNA methylation sites for high-throughput assessment of epigeneticdrug-induced effects in cancer cells. Exp Cell Res 2010; 316:3150-60;Gertych A, et al. (2010). Homogeneity assessment of cell populations forhigh-content screening platforms. In: Information Technology inBiomedicine. Vol. 2. Advances in intelligent and soft computing, Vol.69. Ewa Pietka and Jacek Kawa, Editors, Springer Verlag, Heidelberg,Germany; and Tajbakhsh J, (2012). 3-D Quantitative DNA MethylationImaging for Chromatin Texture Analysis in Pharmacoepigenomics andToxicoepigenomics. In Epigenomics: From Chromatin Biology toTherapeutics. K. Appasani, editor. Cambridge University Press,Cambridge, United Kingdom, each of which is incorporated herein byreference as though fully set forth.

In some embodiments, the image analysis tool operates in three steps: 1)all cells (within imaged populations) are processed for 3D segmentation;2) fluorescence signal residing within the nuclei are measured for (a)determining the 5-methylcytosine load of the entire nucleus, (b) for thegeneration of codistribution maps (scatter plots) of 5mC signals andglobal nuclear DNA (visualized by DAPI), and c) for thevariability/heterogeneity regarding the two first 5mC features.Similarity analysis is conducted of DNA methylation load and created 2Ddiagrams among all cells within each specimen, and cell populationhomogeneity is determined.

With respect to similarity analysis, commonly applied similaritymeasures can be organized into three groups according to objectrepresentation: (a) point-based, including Euclidean and Minkowskidistances, (b) set-based including Jaccard's, Tanimoto's, and Dice'sindices, and (c) probabilistic with Bhattacharyya, Kullback-Leibler's,and correlation-based Mahalanobis distances, respectively (See Dice L R.Measures of the amount of ecological association between species. JEcology 1945; 26:297-302; Bhattacharyya A. On a measure of divergencebetween two statistical populations defined by probabilitydistributions. Bull Calcutta Math Soc 1943; 35:99-109; Mahalanobis P C.On the generalized distance in statistics. Proc Nat Inst Scien India1936; 2:49-55; Kullback S, Leibler R A. (1951), “On Information andSufficiency”. Annals of Mathematical Statistics 22 (1): 79-86; JaccardP. (1912), “The distribution of the flora in the alpine zone”, NewPhytologist 11: 37-50; Rogers D J, Tanimoto T T. (1960), “A ComputerProgram for Classifying Plants”. Science 132 (3434): 1115-1118; ElenaDeza & Michel Marie Deza (2009) Encyclopedia of Distances, page 94,Springer; Levandowsky M, Winter D. (1971), “Distance between sets”,Nature 234 (5): 34-35, all of which are incorporated herein by referencein their entirety as though fully set forth).

As indicated above, in one non-limiting example, Kullback-Leibler's (KL)divergence measurement, a mathematical operation found very suitable forthe analysis of nuclear targets that have no rigid geometrical shape andposition, can be used (See Gertych A, et al. Automated quantification ofDNA demethylation effects in cells via 3D mapping of nuclear signaturesand population homogeneity assessment. Cytometry A 2009; 75:569-83,which is incorporated herein by reference in its entirety as thoughfully set forth). KL divergence can be applied as a similarity measurebetween the normalized scatter plots of individual nuclei and areference scatter plot to allow intra-population assessment of cells. Tomake the KL-values more descriptive, four soft-qualifiers can beintroduced in the software, defining the similarity degree of a cellversus the entire cell population. These degrees can be associated withparticular ranges of KL divergences such as: similar KL∈[0,0.5), likelysimilar KL∈[0.5,2), unlikely similar KL∈[2,4.5), and dissimilar forKL∈[4.5,∞) (FIG. 3 ).

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described can be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as taught or suggested herein. A variety ofalternatives are mentioned herein. It is to be understood that somepreferred embodiments specifically include one, another, or severalfeatures, while others specifically exclude one, another, or severalfeatures, while still others mitigate a particular feature by inclusionof one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (for example, “such as”) provided withrespect to certain embodiments herein is intended merely to betterilluminate the application and does not pose a limitation on the scopeof the application otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element essential tothe practice of the application.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

In closing, it is to be understood that the embodiments of theapplication disclosed herein are illustrative of the principles of theembodiments of the application. Other modifications that can be employedcan be within the scope of the application. Thus, by way of example, butnot of limitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

What is claimed is:
 1. A method, comprising: obtaining a biologicalsample from a subject, the biological sample including a plurality ofcells; determining a 5-methylcytosine (5mC) content and a global DNA(gDNA) content in a nucleus of each of the plurality of cells;generating and displaying a scatter plotting of the 5mC content andgDNA-content for the nucleus; determining a spatial nuclearco-distribution of the 5mC content and gDNA content in the nucleus ofeach of the plurality of cells; determining that a cell of the pluralityof cells is hypomethylated if: the 5mC content in the nucleus of thecell is lower than a 5mC content from one or more non-cancerous ornon-precancerous reference cells by 25% or more; and/or aregression-line angle of the scatter plotting is smaller than aregression-line angle of a scatter plotting in the one or morenon-cancerous or non-precancerous reference cells, when the gDNA contentdefines x-axis, the 5mC content defines y-axis, and the regression-lineangle is with the x-axis of the scatter plotting; based at least in parton a percentage of determined hypomethylated cells in the plurality ofcells exceeding a threshold, determining that the subject has a highrisk of developing clinically verifiable cancer; and treating thesubject for cancer by (i) administering chemotherapy, (ii) administeringradiation therapy, (iii) performing surgery, or (iv) any combinationthereof.
 2. The method of claim 1, wherein the biological samplecomprises sputum.
 3. The method of claim 2, wherein the sputum comprisesthe plurality of cells.
 4. The method of claim 3, wherein the pluralityof cells is of lung origin.
 5. The method of claim 2, wherein thesubject has a history of smoking cigarettes.
 6. The method of claim 2,wherein the subject does not have a history of smoking cigarettes. 7.The method of claim 1, wherein the 5mC content is determined with amicroscope after the plurality of cells has been subjected to (a)immunofluorescence staining with an antibody specific for 5mC and a dyeto delineate the cytoplasm, and (b) counterstaining with4′,6-diamidino-2-phenylindole (DAPI).
 8. The method of claim 1, whereinthe spatial nuclear co-distribution of 5mC and gDNA is determined with amicroscope after the plurality of cells has been subjected to (a)immunofluorescence staining with an antibody specific for 5mC and a dyeto delineate the cytoplasm, and (b) counterstaining with4′,6-diamidino-2-phenylindole (DAPI).
 9. The method of claim 2, whereinthe biological sample is obtained from a subject by a method comprising:administering hypertonic saline into the subject's respiratory tract;and collecting a quantity of sputum that is expelled from the subject asthe result of inhaling said hypertonic saline.
 10. The method of claim9, wherein the hypertonic saline is administered via a nebulizer. 11.The method of claim 10, wherein the hypertonic saline is 3-5% NaCl. 12.The method of claim 7, wherein the microscope is a confocal scanningmicroscope with a resolution equal to or less than 500 nanometers. 13.The method of claim 1, wherein the plurality of cells for determiningthe hypomethylated cells is a plurality of human respiratory cells. 14.The method of claim 1, further comprising: subjecting the cell toimmunofluorescence staining prior to determining the 5mC content; andthen, subjecting the cell to counterstaining prior to determining thespatial nuclear co-distribution of the 5mC content and gDNA.
 15. Themethod of claim 1, wherein the regression-line angle of the scatterplotting for the cell determined to be hypomethylated is less than 45°.16. The method of claim 15, wherein the regression-line angle of thescatter plotting in the one or more non-cancerous or non-precancerousreference cells is more than 45°.
 17. The method of claim 15, whereinthe regression-line angle of the scatter plotting for the celldetermined to be hypomethylated is less than 20° .
 18. The method ofclaim 1, further comprising: generating a first image of the 5mC contentin the nucleus of each of the plurality of cells by utilizinghigh-resolution confocal scanning microscopy; generating a second imageof the gDNA content in the nucleus of each of the plurality of cells byutilizing high-resolution confocal scanning microscopy; loading imagestacks from the first and second images to a computer system configuredwith image analysis software to extract nuclear 5mC/gDNA patterns; anddisplaying the scatter plotting of the 5mC content and global DNA (gDNA)content for the nucleus on a display based on the extracted nuclear5mC/gDNA patterns.